Solubilized phospholipids for stabilizing nucleic acid polymerases

ABSTRACT

Compositions and methods are provided that relate to solubilized phospholipids and their use in stabilizing nucleic acid polymerases. For example, a phospholipid with a tail containing at least 8 carbons can be solubilized in the presence of an amphipathic molecule.

CROSS REFERENCE

This application claims priority from U.S. Provisional Application No.61/481,437 filed May 2, 2011, herein incorporated by reference, and U.S.Provisional Application No. 61/494,458 filed Jun. 8, 2011.

BACKGROUND

Polymerases may have suboptimal activity associated with their stabilityunder reaction conditions. See for example, Eckert and Kunkel, “TheFidelity of DNA Polymerase and the Polymerases used in the PCR” inPolymerase Chain Reaction I: A Practical Approach, McPherson, et al.,eds., Oxford University Press, New York, N.Y., (1991), pp. 235-244.

Early work reported by Capitani, et al., (Molecular and CellularBiochemistry, 27:137-138 (1979)) described the use ofphosphatidylcholine in the form of vesicles to stimulate the activity ofDNA polymerase-α. U.S. Pat. No. 5,792,612 reported the use ofphospholipids in a suspension or matrix in the form of unilamellarliposomes which are combined with polymerases and DNA or RNA to increasethe efficiency of polymerase-dependent amplification reactions. Theunilamellar liposomes were prepared by extrusion through 0.1 μMpolycarbonate membranes with a LiposoFast matrix homogenizer as reportedby MacDonald, et al., (Biochimica et Biophysica Acta-Biomembranes,1061:297-303 (1991)) and were added prior to initiation of the reaction.

One of the limitations of using long-chain phosphatidylcholinephospholipids relates to their insolubility, which necessitates the useof an extruder to create pre-formed vesicles or liposomes. Anotherrelated limitation is that vesicles and liposomes have adverse effectson certain physical attributes such as light scattering in activityassays.

Polymerases for use in polymerase-dependent amplification havereportedly benefited from the stabilizing effect of non-phospholipiddetergents characterized by 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (U.S. Pat. Nos. 6,881,559and 7,429,468). Later-filed applications claim CHAPS under the broaderdescription of zwitterionic detergents for use in DNA amplification(International Publication Nos. WO 2008/013885; WO 2008/077017; U.S.Publication No. 2010/0099150).

Other agents used for polymerase stabilization have also been described.U.S. Pat. No. 6,242,235 describes the use of polyethoxylated surfactantswith polymerases and International Publication No. WO 2008/013885similarly describes a polymerase with a surfactant which “has arelatively small hydrophobic head and two long hydrophilic ethyleneoxide tails that do not form micelles.” The use of non-ionic polymericdetergents and anionic detergents are described in U.S. Pat. Nos.6,127,155 and 7,846,703 and U.S. Publication Nos. 2010/0159528

There is a continuing need to refine and improve stabilizers forpolymerases so as to enhance the activity of the polymerases understorage and reaction conditions.

SUMMARY

In one aspect, a composition includes a polymerase in a buffer, wherethe polymerase is stabilized by a solubilized long-chain phospholipid inwhich the phospholipid comprises a tail with a carbon backbone of atleast 8 carbons.

Various embodiments include one or more of the following features:

-   -   the solubilized long chain phospholipid is a phosphatidylcholine    -   the composition includes a solubilizer such as an amphipathic        molecule such as a lipid having a tail with a carbon backbone of        no more than 8 carbons;    -   the polymerase is a thermostable polymerase;    -   the activity of the polymerase is greater in the presence of the        solubilized phospholipid than in the absence of the solubilized        phospholipid as can be measured in a replication assay by        incorporation of deoxynucleoside triphosphate (dNTP).

The enhancement in polymerase activity in the presence of thesolubilized phospholipid is at least 3-fold, for example at least5-fold.

In a further aspect, a method includes stabilizing a polymerase in abuffer by adding a solubilized long-chain phospholipid in which thesolubilized phospholipid has a tail with at least 8 carbons.

Various embodiments may include:

adding a solubilizer such as a lipid having one or more hydrophobictails where the tail or tails have no more than 8 carbons forming thebackbone of the tail; or obtaining an enhancement of the activity of thepolymerase.

In a further aspect, a kit includes a polymerase in a buffer containinga solubilized phospholipid having at least one or more tails with abackbone of at least 8 carbons forming the tail and a stabilizer.

In a further aspect, a composition, is provided that includes asolubilized long-chain phospholipid in which the phospholipid has atleast one hydrophobic tail containing at least 8 carbons

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C, 2A-C, 3A-C, 4A-C, 5A-C, 6A-C, 7A-C and 8A-C described belowshow enhanced polymerase activity in a primer extension assay that wasdeveloped to measure the effect of solubilized long chain phospholipidson polymerase stability and activity. The embodiment of the assay uses:a serial dilution of Taq DNA polymerase (1.0-0.008 units per reaction ina 2-fold serial dilution); various percentage concentrations ofspecified solubilized long-chain phospholipids (0.3%, 0.1%, 0.025%,0.005% and 0% detergent); M13 DNA; M13 primer; and labeled dNTPs. Theresults show an increase in dNTP-incorporation into a growing DNApolymer due to extension of the annealed M13 primer with time. Thesereadings give a direct measure of polymerase activity.

The plots show that Taq DNA polymerase was substantially inactive asmeasured by dNTP-incorporation in the absence of the solubilizedlong-chain phospholipids. However, the stability and activity of Taq DNApolymerase was significantly improved when a long-chain phospholipid(having a hydrophobic tail of 8 carbons or greater) was used as astabilizer, and a lipid (having a hydrophobic tail of 8 carbons or less)was used as a solvent. The greater the amount of long-chainphospholipid, the steeper the curve in the graph.

FIG. 1A shows the results using1,2-dihexanoyl-sn-glycero-3-phosphocholine (diC₆PC) as a solubilizer and1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC₉PC) as a stabilizer.

FIGS. 1B and 1C shows the structure of the solubilizer and stabilizerrespectively.

FIG. 2A shows the results of using diC₆PC as a solubilizer and1,2-didecanoyl-sn-glycero-3-phosphocholine (diC₁₀PC) as a stabilizer.

FIGS. 2B and 2C shows the structure of the solubilizer and stabilizerrespectively.

FIG. 3A shows the results of using diC₆PC as a solubilizer and1,2-diundecanoyl-sn-glycero-3-phosphocholine (diC₁₁PC) as a stabilizer.

FIGS. 3B and 3C shows the structure of the solubilizer and stabilizerrespectively.

FIG. 4A shows the results of using1,2-diheptanoyl-sn-glycero-3-phosphocholine (diC₇PC) as a solubilizerand diC₁₀PC as a stabilizer.

FIGS. 4B and 4C shows the structure of the solubilizer and stabilizerrespectively.

FIG. 5A shows the results of using1,2-diheptanoyl-sn-glycero-3-phosphocholine (diC₇PC) as a solvent and anether lipid 1,2-di-O-dodecyl-sn-glycero-3-phosphocholine (diOC₁₂PC) as astabilizer.

FIGS. 5B and 5C shows the structure of the solubilizer and stabilizerrespectively.

FIG. 6A shows the results of using a mono-substitutedphosphatidylcholine 1-octanoyl-2-hydroxy-sn-glycero-3-phosphocholine(C₈PC) as a solubilizer and di-substituted phosphatidylcholine (diC₁₀PC)as a stabilizer.

FIGS. 6B and 6C shows the structure of the solubilizer and stabilizerrespectively.

FIG. 7A shows the results of using a phosphorylcholineO-(octylphosphoryl)choline (OPC) as a solubilizer and a diC₁₀PC as astabilizer.

FIGS. 7B and 7C shows the structure of the solubilizer and stabilizerrespectively.

FIG. 8A shows the results of using a phosphorylcholineO-(decylphosphoryl)choline (DPC) as a solubilizer and a diC₁₀PC as astabilizer.

FIGS. 8B and 8C shows the structure of the solubilizer and stabilizerrespectively.

FIG. 9A shows a phospholipids core structure.

FIGS. 9B and 9C show R₁, R₂ and R₃ of the structure shown in FIG. 9A forstabilizers n≧8 and solubilizers n≦8. FIG. 9B shows R₁ and R₂ could beany of the following: —C_(n)H_(2n+1), —C_(n)H_(2n−1), —C_(n)H_(2n−3),—C_(n)H_(2n−5), —C_(n)H_(2n−7), —C_(n)H_(2n−9). FIG. 9C shows R₃ couldbe any of the following: —C₅H₁₃N, —H, —C₂H₇N, —C₃H₇O₂, —C₃H₆O₂ and—C₆H₁₁O₅.

FIG. 10A shows a triglycerides core structure.

FIG. 10B shows R₁, R₂ and R₃, of the structure shown in FIG. 10A forstabilizers n≧4, could be any of the following: —C_(n)H_(2n+1),—C_(n)H_(2n−1), —C_(n)H_(2n−3), —C_(n)H_(2n−5), —C_(n)H_(2n−7),—C_(n)H_(2n−9).

FIG. 11A shows an ether lipids core structure.

FIGS. 11B and 11C show R₁, R₂ and R₃ of the structure shown in FIG. 11Afor stabilizers n≧8 and solubilizers n≦8.

FIG. 11B shows R₁ and R₂ could be any of the following: —C_(n)H_(2n+1),—C_(n)H_(2n−1), —C_(n)H_(2n−3), —C_(n)H_(2n−5), —C_(n)H_(2n−7),—C_(n)H_(2n−9).

FIG. 11C shows R₃ could be any of the following: —C₅H₁₃N, —H, —C₂H₇N,—C₃H₇O₂, —C₃H₆O₂ and —C₆H₁₁O₅.

FIG. 12A shows a sphingolipids core structure.

FIGS. 12B and 12C show R₁, R₂ and R₃ of the structure shown in FIG. 12Afor stabilizers n≧8 and solubilizers n≦8. FIG. 12B shows R₁ and R₂ couldbe any of the following: —C_(n)H_(2n+1), —C_(n)H_(2n−1), —C_(n)H_(2n−3),—C_(n)H_(2n−5), —C_(n)H_(2n−7), —C_(n)H_(2n−9). FIG. 12C shows R₃ couldbe any of the following: —PO₃C₅H₁₃N, —H, —PO₃C₂H₇N, —PO₃C₃H₇O₂,—PO₃C₃H₆O₂, —PO₃C₆H₁₁O₅, —P_(n)O_(3n)H, —C_(6n)H_(10n+1)O_(5n),—C_(6n)H_(10n)O_(5n), —C_(6n)H_(10n-1)O_(5n), —C_(6n)H_(10n-2)O_(5n),—C_(6n)H_(10n-3)O_(5n), —C_(6n)H_(10n+7n)O_(5n+3n)N_(n).

FIG. 13A shows a plasminogens core structure.

FIGS. 13B and 13C show R₁, R₂ and R₃ of the structure shown in FIG. 13Afor stabilizers n≧8 and solubilizers n≦8. FIG. 13B shows R₁ and R₂ couldbe any of the following: —C_(n)H_(2n+1), —C_(n)H_(2n−1), —C_(n)H_(2n−3),—C_(n)H_(2n−5), —C_(n)H_(2n−7), —C_(n)H_(2n−9). FIG. 13C shows R3 couldbe any of the following: —C₅H₁₃N, —H, —C₂H₇N, —C₃H₇O₂, —C₃H₆O₂ and—C₆H₁₁O₅.

FIG. 14A shows the structure of 1,2diphytanoyl-sn-glycero-3-phosphocholine suitable for use as astabilizer.

FIG. 14B shows the structure of L-α-phosphatidylcholine suitable for useas a stabilizer.

FIG. 14C shows the structure of hydrogenated L-α-phosphatidylcholinesuitable for use as a stabilizer.

FIG. 14D shows the structure of 1,2,dioctanoyl-sn-glycero-3-phosphocholine suitable for use as asolubilizer.

FIG. 14E shows the structure of1,2-dinonanoyl-sn-glycero-3-phosphocholine suitable for use as asolubilizer.

DETAILED DESCRIPTION OF EMBODIMENTS

In embodiments of the invention, the problem of solubilization of a longchain phospholipid has been overcome making them suitable as stabilizersof polymerases in storage buffers and reaction buffers. The activity ofsolubilized long-chain phospholipids described herein was determined toreverse loss of activity as might occur during storage of the polymeraseat −20° C. and also under reaction conditions as determined by measuringdNTP-incorporation.

Lack of solubility of the long-chain phospholipids that previouslynecessitated the formation of liposomes or vesicles (U.S. Pat. No.5,792,612) was a deterrent for their use. This problem has been resolvedhere by adding one or more amphipathic molecules of an effective size toenable the long-chain phospholipid to remain in solution as asolubilizer of the otherwise insoluble long-chain phospholipid.

In all instances in which a solubilized phospholipid stabilizer has beentested, it has been found to perform efficiently and effectively fornucleic acid polymerase stability and activity.

A solubilized phospholipid stabilizer described herein is characterizedby a hydrophilic head and at least one hydrophobic tail. It has beendemonstrated that phospholipids having at least one tail with a carbonbackbone of varying numbers of carbons preferably at least 8 carbons canact as stabilizers for nucleic acid polymerases although these moleculeson their own are relatively insoluble in an aqueous buffer. According tothe present embodiments, these molecules can be solubilized by addingone or more amphipathic molecules to the buffer.

Examples of stabilizers include phospholipids with at least one tailcontaining 8-30 carbons, e.g., 8-20 carbons, 8-16 carbons, or 8-12carbons. These include the following: phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylglycerol,phosphatidylserine, and phosphatidylinositol; ether lipids such as1,2-di-O-dodecyl-sn-glycero-3-phosphocholine; sphingolipids such assphingosines, ceramides, sphingomyelin, gangliosides,glycosphingolipids, phosphosphingolipids, and phytosphingosine; anddiacyl glycerols. Diacyl phospholipids with fatty acid chain lengths inthe 8-30 carbon range tend to be insoluble without a suitablesolubilizing agent. Additional examples of stabilizers having at least a8 carbon chain include diC₉PC, diC₁₀PC, diC₁₁PC or diCO₁₂PC (FIGS. 1A-C,2A-C and 3A-C) or diC₁₀PC. These are solubilized when combined with oneor more amphipathic molecules exemplified by a lipid with no more than 8carbons in the lipid carbon chain e.g., diC₆PC, diC₇PC, C₈PC, OPC andDPC (FIGS. 4A-B, 5A-B, 6A-B, 7A-B and 8A-B). Examples of core structuresof stabilizers and solubilizers and examples of R groups are shown inFIGS. 9A-C, 10A-B, 11A-C, 12A-C and 13A-C.

Examples of solubilizing agents include amphipathic molecules such aslipids with one or two tails each containing 3-8 carbons. The lipid mayoptionally include one or more hydrophilic side chains. Examples ofalternative solubilizing agents include phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylglycerol,phosphatidylserine, phosphatidylinositol; ether lipids such as1,2-di-O-dodecyl-sn-glycero-3-phosphocholine; sphingolipids such assphingosines, ceramides, sphingomyelin, gangliosides,glycosphingolipids, phosphosphingolipids, and phytosphingosine; anddiacyl glycerols.

Additional solubilizing agents include anionic, cationic, zwitterionic,or non-ionic amphipathic molecules, for example, Triton® X-100 (UnionCarbide Corp., Midland, Mich.), Tween®-20 (Uniqema Americas, LLC,Wilmington, Del.), IGEPAL® CA-630 (Rhodia Operations, Aubervilliers,France), Triton X-200 (obtainable from Sigma-Aldrich, St. Louis, Mo.),CHAPS, Octyl-β-glucoside, Octyl-α-glucoside, Decyl-β-D-maltoside,MEGA-8, ASB-C80, 3-(N,N-dimethyloctylammonio)propanesulfonate,Heptyl-β-D-1-thioglucoside, Sodium deoxycholate, Brij® 58 (UniqemaAmericas LLC, Wilmington, Del., product obtained from Sigma-Aldrich, St.Louis, Mo.),3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate(CHAPSO),3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate,Nonidet P-40 (obtained from Sigma-Aldrich, St. Louis, Mo.), Teepol 610 S(obtained from Sigma-Aldrich, St. Louis, Mo.),3α,7β-Dihydroxy-5β-cholan-24-oic acid 5β-Cholan-24-oic acid-3α,7β-diol7β-Hydroxylithocholic acid, Taurodeoxycholic acid sodium salt hydrate,Docusate sodium salt, N-Dodecanoyl-N-methylglycine,(Di-isobutylphenoxyethoxyethyl)dimethylbenzylammonium chloride,Pluronic® F-68 (BASF Corporation, Mount Olive, N.J., product obtainedfrom Sigma-Aldrich, St. Louis, Mo.),N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, Cocamidopropylbetaine, Lauramidopropyl betaine, Cholic Acid, Taurocholic Acid, TritonX-301 (Sigma-Aldrich, St. Louis, Mo.), Triton W-30 (Sigma-Aldrich, St.Louis, Mo.), Alkylether hydroxypropyl sultaine,Bis-(2-hydroxyethyl)isodecyloxypropylamine, and Cetylpyridiniumchloride, Poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl etherpotassium salt, Poly(ethylene glycol)monolaurate, Polyoxyethylene (150)dinonylphenyl ether and Nonyl nonoxynol-15 phosphate.

A single solubilizer or a plurality of solubilizers may be used togetherwith a single stabilizer or a plurality of stabilizers in order tostabilize the polymerase in a storage buffer or as needed in a reactionbuffer. A ratio of solubilizer to stabilizer may include 1:1 to 10:1molar ratios with a concentration of solubilizer of 0.001% w/v to 0.5%w/v in the final enzyme reaction buffer. The effective ratio ofsolubilized stabilizer can readily be determined for differentpolymerases using the screening assays described in the examples belowor other assays known in the art which measure polynucleotideamplification products.

Solubilized phospholipid have a stabilizing effect on a wide range ofnucleic acid polymerases including DNA polymerases and thermostablenucleic acid polymerases. As used herein, “nucleic acid polymerase” or“polymerase” refers to an enzyme that catalyzes the polymerization ofnucleotides. Generally, the enzyme will initiate synthesis at the 3′-endof the primer annealed to a nucleic acid template sequence, and willproceed in the 5′-direction along the template. “DNA polymerase”catalyzes the polymerization of deoxynucleotides. DNA polymerases andtheir properties are described in detail in, among other places,Kornberg, et al., DNA Replication, 2nd edition, W.H. Freeman, New York,N.Y. (1991) (Lundberg, et al., Gene, 108(1):1-6 (1991), provided byStratagene, La Jolla, Calif., USA; Hinnisdaels, et al., Biotechniques20:186-8 (1996); Myers and Gelfand, Biochemistry, 30:7661 (1991);Stenesh and McGowan, Biochimica et Biophysica Acta, 475:32 (1977); Diazand Sabino, 1998. Brazilian Journal of Medical and Biological Research,31:1239 (1998); Chien, et al., Journal of Bacteriology, 127:1550 (1976);Takagi, et al., 1997, Applied and Environmental Microbiology, 63:4504(1997); International Publication No. WO 2001/32887; Lecomte andDoubleday, Nucleic Acids Research, 11:7505 (1983); Nordstrom, et al.,Journal of Biological Chemistry, 256:3112 (1981); Cann, et al., Proc.Natl. Acad. Sci. USA, 95:14250-5 (1998)).

Examples of such DNA polymerases include bacterial polymerases such as:Taq DNA polymerase, Hemo KlenTaq® (Wayne M. Barnes, Chesterfield, Mo.)DNA polymerase, KlenTaq DNA polymerase, Aquifex aeolicus (Aae) DNApolymerase, E. coli DNA polymerase I, Klenow Fragment DNA polymerase,Bacillus stearothermophilus (Bst) DNA polymerase, large fragment, BstDNA polymerase I, Bacillus caldotenax (Bca) DNA polymerase, Manta DNApolymerase, Thermus thermophilus (Tth) DNA polymerase, Bacillus smithii(Bsm) DNA polymerase, Thermus brockianus DNA polymerase; Thermotogamaritima (Tma) DNA polymerase; Archaeal DNA polymerases such as 9° N DNApolymerase such as Therminator™ (New England Biolabs, Inc., Ipswich,Mass.); Therminator gamma; Therminator II DNA polymerase, TherminatorIII DNA polymerase; Vent DNA polymerase (New England Biolabs, Inc.,Ipswich, Mass.); Deep Vent™ DNA polymerase (New England Biolabs, Inc.,Ipswich, Mass.); Pfu DNA polymerase; PfuCx DNA polymerase; Phusion® DNApolymerase (Finnzymes, now part of ThermoFisher Scientific, Waltham,Mass.); Phire® DNA polymerase (Finnzymes); Pwo DNA polymerase; KOD DNApolymerase; Neq DNA polymerase; Sulfolobus DNA polymerase IV; Pyrococcuswoesei (Pwo) DNA polymerase (Roche Molecular Biochemicals, Indianapolis,Ind.), JDF-3 DNA polymerase (from Thermococcus sp. JDF-3; archaealDP1/DP2 DNA polymerase II; viral polymerases such as T4 DNA polymerase;T7 DNA polymerase; Phi29 DNA polymerase; and reverse transcriptases suchas Avian Myeloblastosis Virus (AMV) reverse transcriptase; MoloneyMurine Leukemia Virus (MMLV) reverse transcriptase and Thermotogalereverse transcriptase.

Examples of mixtures of polymerase that may be stabilized include: TaqDNA polymerase and Deep Vent DNA polymerase; Taq DNA polymerase and VentDNA polymerase; Thermus brockianus DNA polymerase and Deep Vent DNApolymerase; Thermus brockianus DNA polymerase and Vent DNA polymeraseand Taq DNA polymerase and Vent (exo-) DNA polymerase; and Taq DNApolymerase and Phusion DNA polymerase.

Thermostable DNA polymerases that may be used in embodiments of theinvention include, but are not necessarily limited to, the above recitedpolymerases and mutants, variants and derivatives thereof (see, forexample, U.S. Pat. Nos. 5,436,149; 4,889,818; 5,079,352; 5,614,365;5,374,553; 5,270,179; 5,047,342; and 5,512,462; InternationalPublication Nos. WO 92/06188; WO 92/06200; and WO 96/10640; and Barnes,Gene, 112:29-35 (1992); Lawyer, et al., PCR Meth. Appl., 2:275-287(1993); and Flaman, et al., Nucleic Acids Research, 22(15):3259-3260(1994)).

DNA polymerases or mutants or variants thereof may be stabilized singlyor in mixtures using the stabilizers described herein. These areexemplified below:

In one embodiment, the thermostable DNA polymerase is a Pfu DNApolymerase with a mutation at position V93, wherein the polymerase isexonuclease deficient (e.g. Deep Vent (exo⁻), Vent (exo⁻), Pfu V93(exo⁻)). Methods of making and using Pfu V93 exo⁻ DNA polymerase aredescribed in U.S. Publication No. US 2004-0091873. In anotherembodiment, the polymerase is a fusion protein having polymeraseactivity such as Pfu-Sso7 DNA polymerase, Vent-Sso7 DNA polymerase orDeep Vent-Sso7 DNA polymerase (U.S. Publication Nos.: 2007-0141591,2005-0048530 and U.S. Pat. Nos. 7,666,645 and 7,541,170).

DNA polymerases may be stored in storage buffers either at 1× or athigher concentrations such as 10× for extended periods of time at roomtemperature or below room temperature. DNA polymerases may be introducedinto a reaction mixture for purposes, for example, of amplification.These reaction mixtures may be used at high temperatures necessitated bythe amplification protocol for variable but relatively short periods oftime. The phospholipid stabilizer and solubilizer can be provided as aconcentrated stock for use after dilution. For example, it may beprovided at a 10× concentration in a 10× stock reaction buffer that issuitable for performing a nucleic acid amplification reaction. The 10×stock is diluted to a final 1× working concentration.

Examples of standard storage buffers for polymerases into which thepresent embodiments can be added contain 10 mM Tris-HCl, 100 mM KCl, 1mM Dithiothreitol, 0.1 mM EDTA, and 50% Glycerol.

Examples of typical reaction buffers into which the present mix ofsolubilizer and stabilizer may be added include Taq standard buffer,Thermopol™ detergent-free buffers and detergent-free Phusion HF or GCbuffer (New England Biolabs, Inc., Ipswich, Mass.). The reactionmixtures may include one or more of the following: primers, probes,labeled nucleotides and one or more reagents for labeling the DNA.DNA-labeling reagents include fluorophores, radiolabels and chemicalmoieties. DNA-labeling reagents may further include dual labels,Fluorescence Resonance Energy Transfer (FRET), the use of quenchers (seeU.S. Publication No. 2008-0064071), and intercalating dyes or DNAbinding dyes. The labeling reagents may be introduced into a storagebuffer of a reaction mixture before use or into the reaction bufferafter the reaction (e.g., amplification) has occurred.

The solubilized phospholipid stabilizer of DNA polymerases can be usedfor any biological reaction in vitro for which DNA polymerases are used.Some of these applications are described in U.S. Publication No.2008-0064071 and further include sequencing, cloning and DNA repair. Acommon use of DNA polymerases is in DNA amplification. Solubilizedphospholipid stabilizers have been shown here to be effective atstabilizing a polymerase so as to enhance yields obtained duringamplification. Examples of amplification reactions include thermocyclingamplification reactions such as qPCR, PCR, RT-PCR, Direct PCR, PCR inthe presence of inhibitors (e.g. hemoglobin, humic acid), Emulsion PCRand Bridge PCR; and isothermal amplification including loop-mediatedisothermal amplification (LAMP), amplification such as bioluminescentassay in real time (BART)-LAMP and reverse transcriptase (RT)-LAMP;helicase-dependent amplification (HDA) including RT-HDA,strand-displacement amplification, rolling circle amplification,multiple-displacement amplification, recombinase polymeraseamplification (RPA) and RT-RPA (Kim, et al., Bioanalysis 3(2):227-39(2001); Gill, et al., Nucleic Acids Research, 27(3):224-43 (2008) asappropriate.

An embodiment of the invention includes a kit that comprises a packageunit having one or more containers of the composition, and in someembodiments, includes containers of various reagents used forpolynucleotide synthesis, including synthesis in PCR, sequencing,mutagenesis, and the like. Among other things, the kit may also containone or more of the following items: polynucleotide precursors (e.g.,nucleoside triphosphates), primers, probes, buffers, instructions,labeled nucleotides, intercalating dyes, and control reagents. The kitmay include containers of reagents mixed together in suitableproportions for performing the methods in accordance with the invention.Reagent containers preferably contain reagents in unit quantities thatobviate measuring steps when performing the subject methods. Oneexemplary kit according to embodiments also contains a DNA yieldstandard for the quantitation of the PCR product yields from a stainedgel.

In one embodiment, the kit includes a master mix reagent comprising athermostable polymerase, a phospholipid stabilizer/solubilizer, andpolynucleotide precursors. In another embodiment, the kit includes astorage and/or reaction buffer having a polymerase and at least onephospholipid stabilizer/solubilizer. The storage buffer does not containa detectable label in some configurations. A combination of two or morephospholipid stabilizers and one or more solubilizers may be provided.In yet another embodiment, the kits may further include a separatecontainer having dNTPs. In another embodiment, any of the above kits mayfurther include a separate container having a detectable label.

In an embodiment, the invention is directed to a kit which includes apurified polymerase, at least one phospholipid having a tail containingat least 8 carbons, an amphipathic solubilizer, polynucleotideprecursors, and a labeled nucleotide. In yet another embodiment, theinvention is directed to a kit which includes a purified polymerase, atleast one phospholipid having greater than 8 carbons, an amphipathicsolubilizer, polynucleotide precursors, and a DNA binding dye.

All references cited herein are incorporated in their entirety.

EXAMPLES

Enhancement of stability of a thermostable DNA polymerase (Taq) by thepresence of stabilizer/solubilizer mixtures was investigated. Assayswere developed for determining the reaction yield in primer extensionassays, with or without the presence of phospholipids (obtained fromAvanti Polar Lipids, Inc., Alabaster, Ala.), and amphipathic molecules(also from Avanti Polar Lipids, Inc.) after the polymerase was subjectedto temperatures, which would be expected to inactivate the enzyme in theabsence of non-ionic detergents.

Extension assays were performed in a 20-μl reaction mixture containingM13mp18 single-stranded DNA (6.7 nM) and a 20-fold excess of −47sequencing primer (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) (SEQ ID NO:1). Assaysalso contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 200μM dNTPs. The Taq DNA polymerase was added to give the finalconcentrations shown in the figures. Taq DNA polymerase (800 U/μl) wasstored in concentrated amounts in a detergent-free buffer (10 mMTris-HCl (pH 7.4), 100 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, and 50%glycerol). Prior to the assay, Taq DNA polymerase was diluted in storagebuffer containing 3%, 1%, 0.025%, or 0.05% of each component of a lipidmixture. The enzyme was then added to the final reaction mixture in a1/10 dilution. Standard reactions were also conducted in parallel in theabsence of any lipids. Reactions were set up on ice, and then incubatedin a thermal cycler (Applied Biosystems, now part of Life Technologies,Carlsbad, Calif.). Each reaction mixture, with or without lipids, wasfirst heated at 94° C. for 2 minutes, then 72° C. for 10 minutes. Theyield of reaction product was determined by including [3H]-dTTP (0.65Ci/mmole, Perkin Elmer, Waltham, Mass.) in the reaction mixtures.Reaction products were spotted on DE81 filter disks (Whatman, part of GEHealthcare, Waukesha, Wis.) that were washed three times in 250 mMsodium acetate to remove unincorporated nucleotides. The radioactiveincorporation on filters was determined by scintillation counting tocalculate product yield.

The results are shown in FIGS. 1A-C, 2A-B, 3A-B, 4A-B, 5A-B, 6A-B, 7A-Band 8A-B. It was concluded that a lipid mixture stabilizes thepolymerase and/or enhances polymerase activity if (a) the polymerase isstored in a buffer containing the lipid mixture at storage temperaturesof typically −20° C., (b) the lipid mixture is added to the storagebuffer at any time prior to the assay, and/or (c) the lipid mixture isadded to the reaction mixture.

What is claimed is:
 1. A composition comprising: a thermostablepolymerase in a buffer comprising a solubilized long-chain phospholipid,wherein the phospholipid has at least one hydrophobic tail containing 8to 30 carbons.
 2. A composition according to claim 1, further comprisingan amphipathic solubilizer.
 3. A composition according to claim 2,wherein the amphipathic molecule is a lipid having a hydrophobic tailcontaining no more than 8 carbons.
 4. A composition according to claim1, wherein the at least one long-chain phospholipid is aphosphatidylcholine.
 5. A composition according to claim 1, wherein theactivity of the polymerase is enhanced in the presence of thesolubilized phospholipid, capable of being measured in a replicationassay by incorporation of deoxynucleoside triphosphate.
 6. A compositionaccording to claim 5, wherein the enhancement is at least 3-fold.
 7. Akit, comprising: a thermostable polymerase in a buffer containing asolubilized phospholipid having 8 to 30 carbons and a stabilizer.
 8. Akit according to claim 7, wherein the stabilizer is a lipid having ahydrophobic tail containing no more than 8 carbons.
 9. The compositionof claim 1, wherein said thermostable polymerase is a Taq polymerase.10. The composition of claim 1, wherein the phospholipid has at leastone hydrophobic tail containing 8 to 20 carbons.